1. Field of the Invention
The present invention relates generally to detection of a target nucleic acid sequence and specifically to detection of a cell proliferative disorder associated with a hypermutable nucleic acid sequence in a sample.
2. Description of Related Art
Mammalian genomes consist of unique DNA sequences interspersed with moderately and highly repetitive DNA sequences. Gene mapping by meiotic linkage analysis has traditionally been carried out using variations in unique sequence DNA, such as restriction fragment length polymorphisms (Botstein, et al., Am. J. Hum. Genet., 32:314-331, 1980), as genetic markers. Recently, variations in the repetitive sequence elements such as minisatellite or variable number tandem repeat (VNTR) sequences (Jeffreys, et al., Nature, 314:67-73, 1985; Nakamura, et al., Science, 235:1616-1622, 1987), and microsatellite or variable simple sequence motifs (VSSM) (Litt and Luty, Am. J. Hum. Genet, 44:397-401, 1989; Weber and May, Am. J. Hum. Genet., 44:388-396, 1989) have been found to be useful for linkage studies. One advantage to the use of repetitive sequence variations rather than unique sequence variations is the apparently greater number of alleles present in normal populations when compared to restriction fragment length polymorphisms (RFLPs). A second advantage is the ability to readily detect sequence length variations using the polymerase chain reaction to facilitate the rapid and inexpensive analysis of large numbers of DNA samples.
Microsatellite elements consist of simple mono-, di-, or tri- nucleotide sequences where alleles differ by one or more repeat units (Luty, et al., Am.J. Hum. Genet., 46:776-783, 1990; Tautz, et al., Nature, 322:652-656, 1986; Weber and May, Am. J. Hum. Genet., 44:388-396, 1989). Minisatellites, or VNTR sequences, typically have a repeat unit of 20 to several hundred nucleotides and alleles differ by as little as one repeat unit. Among simple sequences, the (TG)n or (CA)n repeat elements have recently proven extremely useful for meiotc mapping since (1) they are abundant in the genome, (2) display a large number of different alleles, and (3) can be rapidly assayed using the polymerase chain reaction (Litt and Luty, Am. J. Hum. Genet., 44:397-401, 1989; Weber and May, Am. J. Hum. Genet., 44:388-396, 1989).
A number of other short sequence motifs have been found in mammalian genomes (Hellman, et al., Gene, 68:93-100, 1988; Knott, et al., Nuc. Acids Res., 14:9215-9216, 1986; Litt and Luty, Am. J. Hum. Genet., 44:397-401, 1989; Milstein, et al., Nuc. Acids Res., 12:6523-6535, 1984; Stoker, et al., Nuc. Acids Res., 13:4613-4621, 1985; Vassart, et al., Science, 233:683-684, 1987; and Vergnaud, Nuc. Acids Res., 17:7623-7630, 1989), and avian genomes (Gyllensten, et al., Nuc. Acids Res., 17:2203-2214, 1989; Longmire, et al., Genomics, 2:14-24, 1988) and are thought to accumulate by DNA slippage during replication (Tautz, et al., Nature, 322:652-656, 1986) or unequal recombination events (Wolff, et al., Genomics, 5:382-384, 1989). Many of these repeat elements display a high degree of genetic variation and, thus, are also useful for meiotic and mitotic mapping.
The VNTR sequence isolated by Jeffreys, (supra) contains an invariant core sequence GGGCAGGAXG which bears some similarities to the chi sequence of phage lambda (Wolff, et al., Genomics, 5:382-384, 1989) and is detected by a restriction fragment of bacteriophage M13 (Vassart, et al., Science, 233:683-684, 1987). Similar repeat elements have been detected by Nakamura, et al. (Science, 235:1616-1622, 1987) and contain a similar, but distinctive, common core unit GGG--GTGGGG. Elements of this type occur within several known gene sequences including the xcex2 globin locus. Similar VNTR elements have been described within the apolipoprotein B (Boerwinkle, et al., Proc. Natl. Acad. Sci. USA, 86:212-216, 1989; Knott, et al., Nuc. Acids Res., 14:9215-9216, 1986) and collagen type II genes (Stoker, et al., Nuc. Acids Res., 13:4613-4621, 1985) and contain a distinct AT-rich motif. Though a physiological function for repetitive elements of this type has not been defined, they have been suggested as potential hot spots for chromosome recombination (DeBustros, et al., Proc. Natl. Acad. Sci. USA, 85:5693-5697, 1988) or elements important for the control of gene expression (Hellman, et al., Gene, 68:93-100, 1988; Milstein, et al., Nuc. Acids Res., 12:6523-6535, 1984).
Microsatellites represent a very common and highly polymorphic class of genetic elements in the human genome. Microsatellite markers containing repeat sequences have been used for primary gene mapping and linkage analysis as described (Weber, et al., Am. J. Human Genet., 44:388, 1989). PCR amplification of these repeats allows rapid assessment for loss of heterozygosity (LOH) and can greatly simplify procedures for mapping tumor suppressor genes (Ruppert, et al., Cancer Res. 53:5093, 1993; van der Riet, et al., Cancer Res., 54:1156, 1994). More recently, microsatellites have been used to identify specific mutations in certain inherited disorders including Huntington""s disease (HD), fragile X chromosome (FX), myotonic dystrophy (MD), spinocerebellar ataxia type I (SCA1), spino-bulbar muscular dystrophy (SBMA) and hereditary dentatorubralpallidoluysian atrophy (DRPLA) (The Huntington""s Disease Collaborative Research Group., Cell, 72:971, 1993; E. J. Kremer, et al., Science, 252:1711, 1991; G. Imbert, et al., Nature Genet., 4:72, 1993); H. T. Orr, et al., Nature Genet., 4:221, 1993); V. Biancalana, et al., Hum. Mol. Genet., 1:255, 1992, M-Y,. Chung, et al., Nature Genet., 5:254, 1993, R. Koide, et al., Nature Genet., 6:9, 1994).
Microsatellite instability has recently been described in human cancers. For example, microsatellite instability has been reported to be an important feature of tumors from hereditary non-polyposis colorectal carcinoma (HNPCC) patients (Peltomxc3xa4ki, et al., Science, 260:810, 1993; Aaltonen, et al., Science, 260:812, 1993; Thibodeau, et al., Science, 260:816, 1993). Moreover, microsatellite instability, demonstrated by expansion or deletion of repeat elements has been reported in colorectal, endometrial, breast, gastric, pancreatic, bladder neoplastic tissues (J. I. Risinger, et al., Cancer Res., 53:5100, 1993; H-J. Han, et al., Cancer Res., 53:5087, 1993; P. Peltomxc3xa4ki, et al., Cancer Res., 53:5853, 1993; M. Gonzalez-Zulueta, et al., Cancer Res., 53:5620, 1993), and recently in SCLC. In HNPCC patients, this genetic instability is due to inherited and somatic mutations of a critical mismatch repair gene (hMSH-2). Mutations of hMLH-1 and other critical mismatch repair genes may also be responsible for the instability detected in HNPCC patients.
Cancer remains a major cause of mortality worldwide, and despite advancements in diagnosis and treatment, the overall survival rate has not improved significantly in the past twenty years. There remains an unfulfilled need for a more sensitive means of early diagnosis. Typical assays to detect rare infiltrating tumor cells in clinical samples utilize amplification methods that require additional cloning steps and synthesis of a large number of oligomer specific probes to detect a wide variety of oncogenic mutations for each tumor type (Sidransky, et al., Science, 252:706, 1991; Sidransky, et al., Science, 256:102, 1992). The present invention provides a sensitive assay to detect a variety of cancers using hypermutable microsatellite markers and an amplification strategy which eliminates the need for additional cloning steps.
The present invention provides a fast, reliable, sensitive screening method for the detection of a cell proliferative disorder in various clinical samples. The invention utilizes amplification of microsatellite nucleic acid (small repeat sequences) to detect a clonal population of cells in a clinical sample.
The invention is based on the unexpected finding that microsatellite alterations are detectable as a clonal population of cells in the DNA of cytologic clinical samples. These samples include urine, sputum, and histopathologic margins obtained from cancer patients.
The invention provides a method for detecting a mammalian cell proliferative disorder (ie., neoplasia) associated with a hypermutable mammalian target nucleic acid in a specimen, comprising isolating the nucleic acid present in the specimen and detecting the presence of the hypermutable target nucleic acid, typically following amplification of the nucleic acid.
In one embodiment, the amplification step in the method of the invention is performed as a multiplex reaction. Therefore, instead of performing multiple amplification reactions to identify each clonal alteration, primers for different markers are combined in one simple amplification reaction, enhancing the identification of a large proportion of cell proliferative disorders.